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Fig. 3 | Skeletal Muscle

Fig. 3

From: Optimized method for extraction of exosomes from human primary muscle cells

Fig. 3

Schema summarizing the protocols used to extract muscle exosomes from the primary human myotube culture medium, using either the ultracentrifugation or the modified polymer-precipitation strategy. For a single data point, 14 flasks of 225 cm2 are plated with 7.5 × 106 myoblasts. After 24 h, once the myoblasts have attached to the flask, they are rinsed 6 times in DMEM and then differentiated into myotubes by cultivating them in DMEM. After 72 hr, the conditioned medium is collected for muscle exosome extraction. After removing dead cells (200 g, 10 min, RT), cell debris (4000 g, 20 min, 4 °C), and ectosomes (20,000 g, 70 min at 4 °C, and filtered at 0.22 μm), the cleared media is subjected to exosome extraction either by the ultracentrifugation protocol or by a modified polymer-precipitation protocol. Ultracentrifugation is at 100,000g (70 min, 4°C), which is followed by washing the pellets three times with PBS (100,000 g, 70 min, 4°C). The subsequent pellet is then either resuspended in 100 μl of PBS or in NuPAGE™ LDS sample buffer for western blot. For the modified polymer-precipitation protocol, the polymer is added at half the volume of the pre-cleared media and incubated overnight at 4 °C. The mix is then centrifuged at 10,000g for 70 min at 4 °C. The subsequent pellet is then washed 3 times in PBS using a 100 kDa Amicon® filter column. Western blot shows the rescue of the epitope CD63 after 3 washes in PBS

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